Comparative analysis of neuroinvasion by Japanese encephalitis virulent and vaccine viral strains in an in vitro model of human blood-brain barrier.

Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.

Role of PDZ-binding motif from West Nile virus NS5 protein on viral replication.

West Nile virus (WNV) is a Flavivirus, which can cause febrile illness in humans that may progress to encephalitis. Like any other obligate intracellular pathogens, Flaviviruses hijack cellular protein functions as a strategy for sustaining their life cycle. Many cellular proteins display globular domain known as PDZ domain that interacts with PDZ-Binding Motifs (PBM) identified in many viral proteins. Thus, cellular PDZ-containing proteins are common targets during viral infection. The non-structural protein 5 (NS5) from WNV provides both RNA cap methyltransferase and RNA polymerase activities and is involved in viral replication but its interactions with host proteins remain poorly known. In this study, we demonstrate that the C-terminal PBM of WNV NS5 recognizes several human PDZ-containing proteins using both in vitro and in cellulo high-throughput methods. Furthermore, we constructed and assayed in cell culture WNV replicons where the PBM within NS5 was mutated. Our results demonstrate that the PBM of WNV NS5 is important in WNV replication. Moreover, we show that knockdown of the PDZ-containing proteins TJP1, PARD3, ARHGAP21 or SHANK2 results in the decrease of WNV replication in cells. Altogether, our data reveal that interactions between the PBM of NS5 and PDZ-containing proteins affect West Nile virus replication.

Innovative in cellulo method as an alternative to in vivo neurovirulence test for the characterization and quality control of human live Yellow Fever virus vaccines: A pilot study.

Live attenuated vaccines have proved to be mostly valuable in the prevention of infectious diseases in humans, especially in developing countries. The safety and potency of vaccine, and the consistency of vaccine batch-to-batch manufacturing, must be proven before being administrated to humans. For now, the tests used to control vaccine safety largely involve animal testing. For live viral vaccines, regulations require suppliers to demonstrate the absence of neurovirulence in animals, principally in non-human primates and mice. In a search to reduce the use of animals and embracing the 3Rs principles (Replacement, Reduction, Refinement in the use of laboratory animals), we developed a new Blood-Brain Barrier Minibrain (BBB-Minibrain) in cellulo device to evaluate the neuroinvasiveness/neurovirulence of live Yellow Fever virus (YFV) vaccines. A pilot study was performed using the features of two distinct YFV strains, with the ultimate goal of proposing a companion test to characterize YFV neurovirulence. Here, we demonstrate that the BBB-Minibrain model is a promising alternative to consider for future replacement of YFV vaccine in vivo neurovirulence testing (see graphical abstract).

Identifying Attenuating Mutations: Tools for a New Vaccine Design against Flaviviruses.

Emerging Flaviviruses pose an increasing threat to global human health. To date, human vaccines against yellow fever virus (YFV), Japanese encephalitis virus (JEV), dengue virus (DV), and tick-borne encephalitis virus (TBEV) exist. However, there is no human vaccine against other Flaviviruses such as Zika virus (ZIKV) and West Nile virus (WNV). In order to restrict their spread and to protect populations against the diseases they induce, vaccines against these emerging viruses must be designed. Obtaining new live attenuated Flavivirus vaccines using molecular biology methods is now possible. Molecular infectious clones of the parental viruses are relatively easy to generate. Key mutations present in live attenuated vaccines or mutations known to have a key role in the Flavivirus life cycle and/or interactions with their hosts can be identified by sequencing, and are then inserted in infectious clones by site-directed mutagenesis. More recently, the use of chimeric viruses and large-scale reencoding and introduction of microRNA target sequences have also been tested. Indeed, a combination of these methods will help in designing new generations of vaccines against emerging and reemerging Flaviviruses.

Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses.

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo, these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N1-acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE: RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex.

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

The E glycoprotein plays an essential role in the high pathogenicity of European-Mediterranean IS98 strain of West Nile virus.

West Nile virus (WNV) is the most widespread arbovirus in the world. Several recent outbreaks and epizootics have been reported in Europe and the Mediterranean basin with increased virulence. In contrast to the well-characterized American and Australian strains, little is known about the virulence determinants of the WNV European-Mediterranean strains. To investigate the viral factors involved in the virulence of these strains, we generated chimeras between the highly neuropathogenic Israel 1998 (IS-98-ST1, IS98) strain and the non-pathogenic Malaysian Kunjin virus (KJMP-502). In vivo analyses in a mouse model of WNV pathogenesis shows that chimeric virus where KJMP-502 E glycoprotein was replaced by that of IS98 is neuropathogenic, demonstrating that this protein is a major virulence determinant. Presence of the N-glycosylation site had limited impact on virus virulence and the 5'UTR does not seem to influence pathogenesis. Finally, mice inoculated with KJMP-502 virus were protected against lethal IS98 infection.

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts.

UNLABELLED: Japanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuation in vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virus in vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts. IMPORTANCE: JEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.